Lignocellulosic feedstock is a term commonly used to describe plant-derived biomass comprising cellulose, hemicellulose and lignin. Much attention and effort has been applied in recent years to the production of fuels and chemicals, primarily ethanol, from lignocellulosic feedstocks, such as agricultural wastes and forestry wastes, due to their low cost and wide availability. These agricultural and forestry wastes are typically burned or land-filled. Thus, using these lignocellulosic feedstocks for ethanol production offers an attractive alternative to disposal.
The first chemical processing step for converting lignocellulosic feedstock to ethanol, or other fermentation products, involves breaking down the fibrous lignocellulosic material to liberate sugar monomers from the feedstock for conversion to a fermentation product in a subsequent step of fermentation.
There are various known methods for producing fermentable sugars from lignocellulosic feedstocks, the most prominent one involving an acid or alkali pretreatment followed by hydrolysis of cellulose with cellulase enzymes and β-glucosidase. The purpose of the pretreatment is to increase the cellulose surface area and convert the fibrous feedstock to a muddy texture, with limited conversion of the cellulose to glucose. Acid pretreatment typically hydrolyses the hemicellulose component of the feedstock to yield xylose, glucose, galactose, mannose and arabinose and this is thought to improve the accessibility of the cellulose to cellulase enzymes. The cellulase enzymes hydrolyse cellulose to cellobiose which is then hydrolysed to glucose by β-glucosidase. Hydrolysis of the cellulose and hemicellulose can also be achieved with a single-step chemical treatment in which the lignocellulosic feedstock is contacted with a strong acid or alkali under conditions sufficient to hydrolyse both the cellulose and hemicellulose components of the feedstock to sugar monomers.
After production of a stream comprising fermentable sugar from the lignocellulosic feedstock, the sugars are fermented to ethanol or other fermentation products. If glucose is the predominant substrate present, the fermentation is typically carried out with a Saccharomyces spp. yeast that converts this sugar and other hexose sugars present to ethanol. However, glucose can also be fermented to other commercial products including lactic acid, sorbitol, acetic acid, citric acid, ascorbic acid, propanediol, butanediol, xylitol, acetone and butanol. This conversion can be carried out by a variety of organisms, including Saccharomyces spp.
One significant problem with enzymatic hydrolysis processes is the large amount of cellulase enzyme required, which increases the cost of the process. The cost of cellulase accounts for more than 50% of the cost of hydrolysis. There are several factors that contribute to the enzyme requirement, but one of particular significance is the presence of compounds that reduce the reaction rate of cellulases and/or microorganisms in the subsequent fermentation of the sugar. These compounds can also inhibit yeast, which decreases ethanol production and consequently makes the process more costly. Although the effects of inhibitors can be reduced by performing the hydrolysis at a more dilute concentration, this requires the use of a large hydrolysis reactor, which adds to the expense of the process.
One class of inhibitors released during the process are soluble inhibitors such as furfural, hydroxymethyl furfural, furan derivatives, organic acids, such as acetic acid and soluble phenolic compounds derived from lignin. Further examples of soluble inhibitors are glucose and cellobiose, which cause end-product inhibition on cellulases and beta-glucosidase, respectively.
Lignin is another inhibitor present in process streams in either soluble or insoluble form. Various groups have documented the negative effects of lignin on cellulase enzyme systems. Removal of lignin from hardwood (aspen) was shown to increase sugar yield by enzymatic hydrolysis (Kong et al., 1992, Applied Biochemistry and Biotechnology, 34/35:23-25). Similarly, removal of lignin from softwood was shown to improve enzymatic hydrolysis of the cellulose, an effect attributed to improved accessibility of the enzymes to the cellulose (Mooney et al., 1998, Bioresource Technology, 64:113-119). Other groups have demonstrated that cellulases purified from Trichoderma reesei bind to isolated lignin (Chernoglazov et al., 1988, Enzyme and Microbial Technology, 10(8):503-507) and have speculated on the role of the different binding domains in the enzyme-lignin interaction (Palonen et al., 2004, Journal of Biotechnology, 107:65-72). Binding to lignin and inactivation of Trichoderma reesei cellulases has been observed when lignin is added back to a pure cellulase system (Escoffier et al., 1991, Biotechnology and Bioengineering, 38(11):1308-1317).
A variety of methods have been suggested to reduce the negative impact of lignin on cellulases. Non-specific binding proteins (e.g. bovine serum albumin) have been shown to block interactions between cellulases and lignin surfaces (U.S. Publication Nos. 2004/0185542, U.S. Publication No. 2006/088922, WO 2005/024037 and WO 2009/429474). Other chemical blocking agents and surfactants have been shown to have a similar effect (U.S. Pat. Nos. 7,972,826 and 7,354,743). Yet another approach involves designing recombinant cellulases that are resistant to the inhibitory effects of lignin. Recombinant cellulases exhibiting reduced interactions or inactivation by lignin by genetic modification have been reported (WO 2010/096931).
A further approach to reduce the negative impact of lignin on the cellulase system involves removing lignin upstream of cellulase addition. Chang and Holtzapple (2000, Applied Biochemistry and Biotechnology, 84-86:5-37) examined the effects of acetic acid and lignin removal on the digestibility of poplar wood by cellulase enzymes. Cao et al. (1996, Biotechnology Letters, 18(9):1013-1018) disclose a method of steeping corn cobs with 2.9 M ammonium hydroxide for 24 hours at 26° C., which removed 80-90% of the lignin along with almost all the acetate from the feedstock. Organosolv pretreatment is a further method to remove all or a portion of lignin upstream of enzymatic hydrolysis. This pretreatment involves the addition of organic solvents, such as ethanol, to lignocellulosic feedstock in order to extract the lignin.
Despite these efforts, there is a need for a more efficient process that comprises a step of carrying out enzymatic hydrolysis with cellulases. In particular, there is a need in the art to further reduce costs associated with such a process so as to make it more commercially viable.